forARRA years 1-2funding The goals of this project are to define how HIV-1 interacts with pDC and to elucidate the role of pDC cells in HIV-1 replication and pathogenesis. As the major sensor of viral infections, altered pDC level/activity may playa critical role during HIV-1 disease progression. However, the role of pDC cells in F-IIV infection and pathogenesis is poorly understood, mainly due to the lack of robust in vivo models. The DKO-hu I-4SC model is ideal for this purpose. With a stable functional human immune system, functional pDC cells are developed in normal proportion in all lymphoid organs in DKO-hu mice. HIV-1 establishes persistent infection, with immune hyperactivation and depletion of human CD4 T cells. We have also shown that, during HIV-1 infection, PDC cells are productively infected, activated, depleted and functionally impaired in DKO-hu HSC mice. HIV-1 with the pathogenic R3A Env also efficiently activates PDC in vitro, correlated with its high binding affinity to CD4 receptor and coreceptors. Based on our preliminary findings and reports from Sly-infected monkeys or HIV- infected patients, postulate that HIV-1 intimately interacts with PDC cells, and chronic engaging of PDC during persistent HIV infection wifl deplete or impair PDC activity. The reduced or altered PDC activity contributes to chronic HIV infection, hyperimmune activation and AIDS progression. :off Cap inM f/1 E-C Q?__. ="O 020 -_a '<a o.- S0mwoo OIL .g. -nd"-0& (nom The following modified specific aims are proposed to test these hypotheses. First, we wil iinvestigate the Induced by HIV infection, by genetically analyzing the candidate signaling pathways (SA2a). Third, we will proliferation and survival of pOC cells during early and late-chronic HIV-1 pDC activation with genetic approaches. In addition, we will also define the signaling defects in pOC cells Second, we will define the role depletes pOC, treat DKO-hu mice with the pOC-specific ILT7 mAb conjugated with the Saporin toxin, which specifically to test the role of pOC during infection (SA3c). of each relevant receptor (C04, CCR5, ... CXCR4, BOCA2, TLR7 and TLR9) in infection in OKO-hu mice (SA1a). proliferation and survival of pDC cells during early and late-chronic HIV-1 infection in DKO-hu mice (SA1a). Second. we will define the role of each relevant receptor (CD4, CCR5, CXCR4, BDCA2, TLR7 and TLR9) in pDC activation with genetic approaches. In addition, we win also define the signaling defects in pDC cells induced by HIV infection, by genetically analyzing the candidate signaling pathways (SA2a). Third, we will [unreadable]-' ANC dmo treat DKO-hu mice with the pDC-specific ILT7 mAb conjugated with the Saporin toxin, which specifically depletes pDC, to test the role of pOC during infection (SA3c). ... C'O' any shy We will thus locus on the most fundamental questions of pDC cells in F-IIV pathogenesis. Elucidation of the will therapeutic strategies. will facilitate not only our understanding of pDC biology in HIV pathogenesis, but also development of novel mechanism therapeutic strategies. We will mechanism by which HIV-1 interacts with pDC cells and their role in HIV-1 infection and AIDS pathogenesis facilitate thus by which not focus only on our HIV-1 the understanding most interacts fundamental with of pOC pOC questions of pOC cells biology 0 and in their role HIV pathogeneSiS, cells in in HIV-1 HIV pathogenesis. infection but also and development of novel AIDS Elucidation pathogeneSiS of the O--_ BSc 8B1I.ModifiedSpecific Aim Section for firsttwoyear ARRAfunding As the major sensor of viral iinfections, altered pODC elevel/activity may pllaya crrititiical rrolle duriing HIVIV-1 disease progress. disease progressioionn.. Howevveer,r,how HIV infection,, activates,, reduces and imimpairs pDOC ccellsls is poorly 0 0 0 N understood. The DKO-hu HSC model is ideal for this purpose. With a stable functional human immune system, functional pDC cells are developed in normal proportion in all lymphoid organs in DKO-hu mice. HIV-1 establishes persistent infection, with immune and depletion of human C04 T cells. Based on our preliminary findings and with POC and BDCA2 on PDC cells and TLR7 in The reduced or altered PDC activity contributes to aberrant hyper.immune activation and AIDS progression. The following 0._ functional pDC cells are developed in normal proportion in all lymphoid organs in DKO-hu mice, HIV-1 establishes persistent infection, with immune hyperactivation and depletion of human CD4 I cells. Based on activity. intimately interacts understood. The OKO-hu HSC model is ideal for this purpose. With a stable functional human immune system, endosomes), and chronic exposure of POC during modified specific aims are proposed for this 2-year ARRA funded ([unreadable]p persistent HIV infection will deplete or impair PDC patients, I postulate that HIV-1 period. 00, N-C our pretiminary findings and reports from Sly-infected monkeys or HIV-infected patients, I postulate that HIV-1 activity. The reduced or altered PDC activity contributes to aberrant hyper-imrnune activation and AIDS TLR7 in endosomes). and chronic exposure of PDC during persistent HIV infection will deplete or impair PDC intimately interacts with PDC cells (via direct binding to CD4, CCR5, CXCR4 and BDCA2 on PDC cells and progression. The following modified specific aims are proposed for this 2-year ARRA funded period. --' reports from SIV-infected monkeys or HIV-infected cells (via direct hyperactivation binding [unreadable]o. to CD4, CCR5, CXCR4 N._ a33 L-- Oft 0'9M (1)Tostudy how HIIV-1-1ininffectiionalters homeostasis and function ofhuman pDoCe cells in vivo: a. Does HIlV-1 ininfefecttioionmodullateprolliiferation ofpDOC cells in lymphoid organs in vivo? We will investigatettheprrolilfieferratitoionppDOCcceellsllsdduurirninggeaeralyrlyanadndlaltaet-ec-hcrhornoicniFc-H11IV-1 infection in DKO-hu miice. W=o ~[unreadable]ai mad 1-6 d<= ELL ram Moo map 0-5 any GUI (2) To investigate how lH-IV-1inintteractswiith poDeCandiimpairs poDeCactivity: a. What is the relative role of pDC receptors in activation of pDC cells? We will define the role of each relevant receptor (CD4, C0R5, CXCR4, BDOCA2, TLR7 and TLR9) in pDC activation with specific inhibitors in PBMC culture and with genetic approaches in the GEN2.2 human pDOC cell. We will define the signaling defects in pDC cells induced by HIVininfefecttiion,bygenetitcicallylyanalylyziningththe candidate signaling pathways. candidate signaling pathways. cell. activation a. What is the relative role of pDC receptors in activation of pDC cells? will We will define with define the specific role the of each inhibitors signaling relevant defects in in PBMC receptor culture pDC ceUs (C04, and induced with CCR5, genetic LL' by CXCR4, 01O duo Esc (3) To define role of pDoeC cells in HIV-1infection and pathogenesis invivo: c. Does depletion of pODC in vivo affect HIV-1 infection and pathogeneSsiiSs? >_n We will treat DKO-hu mice with the pDC-specific mAb conjugated with the Saporin toxin, which specifically depletes pDOC,to test the role ofpDOC during iinfectiion. will t...reat DKO-hu mice with the pOC-specific mAb conjugated with the Saporin toxin, which specifically E.., 5q. 8B2. Modified Specific Aim Section for years 3 and 4 NIAlIODfunding (1) Tostudy how HIV-1iinfectionallters homeostasis and function ofhuman pOoeC cells in vivo: b. Does HIV-1 infection affect pDoCe migration and survivatl in lymphoid organsinin viivo? We will iinvestigate the miigration and surviva'l of pDoCecelllsdudruirninggeaeralyrlaynadnldatel-acther-ocnhirconHicIVH-1IVi-n1feicntfieocntionn in DKO-hu mice. CFO CI=L o.3 (2)Toiinvestigate how HIV-1iintteracttswith pODCand impairs pOCactivity: b.. What genes and signaling pathways are atltered in pIoJeC cells "Wanergized~" by HIV infection in vivo? We wil purify pDoCe cells from mock- or HIV-infected DOKO-hu mice to identify genes that are deregulated by HIV infection. We wil define the signaling defects in pDCe cels inducedby HIV infection. ^-o p-- N.' (3)Todefine role of pOCcelllsin HIV--1iinfection and pathogenesis in vivo: a. Does elevated pDoCe activation affect HIV-1 infection and immuno-pathogenesis? b. Does decreased pDoCe activity viia TLR7f/9 antagonists afect HIV-1 infection and pathogenesiis in vivo? We will study iiffsstitmimulation or ininhibition of pDoCe acttiivation with the agonistic or antatagonistic ligands of TLR7fTLR9 before or during HIV infection will affect HIV replication and immuno-pathogenesis. '<o TLR7/TLR9 before or during HIV infection will affect HIV replication and immuno-pathogenesis. L.. ... Lam. ..J 30c C.